Process for the quantification of cell populations or subpopulations and a reagent suitable therefor

ABSTRACT

A process for quantification of cell populations or subpopulations, by incubating a sample with labelled antibodies directed against characteristic surface antigens of the cell population to be quantified to form labelled antibody/antigen complexes. Standards with known, differing particle concentration and having comparable sedimentation behaviour to the cells to be determined and further, carrying molecules which are directed against the labelled antibody or a part hereof are also incubated with the labelled antibodies. The cells of the sample solution, as well as the particles of the standard solution, are separated off from the excess labelled antibodies and the amount of the labelling is measured not only on the cells but also on the particles. By comparison of the measurement value from the sample with the measurement values from the standard solutions, there is ascertained the number of cells to be determined in the sample. The standard and a reagent kit for carrying out the process are also the subject of this application.

This application is a divisional of Ser. No. 927,803, filed Nov. 5,1986, now U.S. Pat. No. 4,876,189.

The present invention is concerned with a process for the quantificationof cell populations or subpopulations, especially blood cells, as wellas with a reagent for carrying out the process.

The process according to the present invention is especially suitablefor the determination of lymphocytes, in particular T-lymphocytes ortheir subpopulations, the T-helper and T-suppressor cells.

T-lymphocytes have regulatory functions for the humoral as well as forthe cellular immune system. They are classified according todevelopmental stages and function. In the case of T-cells, variousmaturation stages, as well as sub-groups, must be differentiated. Thesecan be characterised by surface antigens, which are specific forindividual developmental stages and for functional sub-groups.Classifications are possible, on the one hand, according to functionalcriteria and, on the other hand, by serological discrimination. By meansof comparisons of the various antigen compositions, conclusions can bedrawn regarding the cell populations and thus regarding the instantimmune state of the donor.

The dormant T-cells must first be stimulated by antigen before they canbe functionally active. Thus, the T-helper cells stimulate theB-lymphocytes to proliferate, to differentiate and to produceantibodies. From the lymphoid cell series, they also activate themacrophages and the T-suppressor cells which are stimulated to thecytotoxic reaction against cells with specific surface antigens.

In the activated state, T-suppressor lymphocytes bring about thesuppression of the antibody production and the reaction of autologousT-cells in the desired lymphocyte culture. The T-suppressor cells alsoshow cytotoxic reaction against the target cells after sensitisationwith HLA antigen-carrying cells (HLA=human leukocyte antigen).

The usual concentrations in the blood, as well as the quotient ofT-helper and T-suppressor cells, lie in the following ranges:

    ______________________________________                                        T-lymphocytes (total)                                                                         0.5-1.5  10.sup.6 cells/ml. blood                             T-suppressor    0.15-0.45                                                                              10.sup.6 cells/ml. blood                             T-helper        0.3-0.9  10.sup.6 cells/ml. blood                             T-helper/T-suppressor                                                                         1.2-3.9                                                       ______________________________________                                    

Various diseases can bring about displacements in the composition of theT-cell subpopulations in which the production of a population is reducedor strengthened. These changes occur very early in the course of thedisease. Such diseases include, for example, diseases of the rheumaticcomplex, autoimmune diseases (e.g. rheumatic arthritis, systemic lupuserythematosus), infectious diseases (e.g. AIDS and viral and fungalinfectious diseases) and malignant T-cell diseases (e.g. leukaemias andlymphomas).

The determination of the immune status, i.e. the totality of theT-lymphocytes and the relative proportion of the T-helper and of theT-suppressor cells, thus provides a considerable diagnostic advance.

The previous methods of determination for cell populations andsubpopulations consist preponderantly in the coloration of the cellswith antibodies of corresponding specificity which are labelled withfluorescent dyestuff and the counting thereof under a fluorescencemicroscope. It is obvious that this is a very laborious andpersonnel-intensive method and only permits a subjective assessment.

This principle can be mechanised and objectivated with the help of a"fluorescent activated cell sorter" (FACS). In this way, the mechanicalcounting can admittedly be omitted. However, the method involves aconsiderable expense for apparatus and requires highly qualifiedpersonnel for the operation.

It is an object of the present invention to provide a simpler methodwith which not only the total number of the cell population but also ofcertain subpopulation can be determined.

Thus, according to the present invention, there is provided a processfor the quantification of cell populations or subpopulations, wherein asample with the cell population to be determined is incubated withlabelled antibodies which are specifically directed againstcharacteristic surface antigens of the cell population to be quantified,one or more standards with known, differing particle concentration areincubated with the same labelled antibodies in the same way, thestandard solution consisting of particles which, in their sedimentationbehaviour, are comparable with the cells to be determined and are loadedwith molecules which are directed against the labelled antibody or apart thereof, the cells of the sample solution, as well as the particlesof the standard solution, are separated off from the excess labelledantibodies, the amount of the labelling is measured not only on thecells but also on the particles and by comparison of the measurementvalue from the sample with the measurement values from the standardsolutions, there is ascertained the number of cells to be determined inthe sample.

The process according to the present invention is generally usable inorder to quantify cell populations and subpopulations, the method beingespecially useful for the quantification of blood cells and particularlyof lymphocytes. The process has proved to be especially advantageous forthe determination of the total number of the T-cells and of the T-helperand T-suppressor subpopulations. Thus, there can be achieved adependable and precise statement regarding the instant immune status forthe diagnosis and control of the course of the therapy.

As antibodies which are directed against antigens, which arecharacteristic for the cell population to be determined (cell surfaceantigens), there can be used not only polyclonal but also monoclonalantibodies. There can be used the complete antibodies or also antibodyfragments. The monoclonal antibodies are especially preferred.

For labelling the antibodies used, various known methods are available.There can be used all conventional labelling agents, for exampleradioisotopes, dyestuffs, fluorescent dyestuffs and enzymes. Accordingto the present invention, labelling with enzymes is especiallypreferred. As labelling enzymes, there can be employed, for example,peroxidase, alkaline phosphatase, glucose oxidase or especiallypreferably β-galactosidase.

The test is preferably carried out as an antibody binding test andpreferably as a solid phase immunoassay. As solid phase, there are usedthe cells to be determined or the particles of the standard whichpossess a size and density or a sedimentation behaviour similar to thecells.

In the development of the test, it was an especial difficulty to find astandard which can imitate the properties of lymphocytes so thatlymphocytes can be replaced by these. The standard is to possess theadvantages of a natural lymphocyte standard without, however, having itsdisadvantages: lymphocytes are not available to the needed extend,lymphocytes are non-uniform, which results in a deficientreproducability from batch to batch and lymphocytes are not sufficientlystable, which gives rise to problems with regard to storage stability ofa lymphocyte standard.

All these disadvantages could be overcome by a standard with artificialparticles.

As such particles are preferred small spheroids which can consist ofglass or synthetic resin. Especially advantageous are synthetic resinparticles, particularly latex particles, which are built up on the basisof methacrylate. These particles should preferably have a diameter of1-20 μm. and especially of 7-13 μm.

On to the standard particles are coupled molecules (proteins) which aredirected against the labelled antibodies or a part thereof. On the onehand, they can be proteins which possess the same antigenic action asthe surface antigens on the cells to be determined. However, theparticles can also carry anti-antibodies which are directed against thelabelled antibodies and preferably against the F_(C) part of thisantibody. Finally, on the particles there can also be present antibodieswhich specifically recognise the labelling, for example, in the case ofan enzyme labelling, the enzyme.

The present invention also provides a standard for the quantification ofcell populations or subpopulations, wherein it contains particles whichare similar in their sedimentation behaviour to the cells to bedetermined and carry molecules which are directed against the labelledantibody or a part hereof.

For the preparation of this standard, the particles are first activated,for example with sodium periodate. Subsequently, a part of the activatedparticles is coupled with a non-specific antibody, for example sheepIgG, according to known methods. The other part of the activatedparticles is linked with the molecule directed specifically against thelabelled antibody or against a part hereof. By mixing variousproportions of these two differently labelled particle types, standardsolutions are produced which are constant with regard to their particlenumber but vary with regard to the bound specific molecules. The exactconcentration of specific molecules in a particular standard can bedetermined by comparison with a known lymphocyte concentration which hasbeen determined by conventional counting out using immune fluorescenceor with the help of a cell sorter. The binding ability of the socalibrated particles corresponds to a definite number of the cellpopulation or subpopulation to be determined.

The standard can be present as a solution or as a lyophilisate. Beforeuse, the lyophilisate must be resuspended in an appropriate solvent, forexample double distilled water.

The present invention also provides a reagent mixture for carrying outthe process according to the present invention, wherein it contains thelabelled antibody which is specifically directed against the cellpopulation to be determined, one or more standards, an appropriatedetection system for the measurement of the labelling agent, as well asfurther adjuvants possibly necessary.

The individual components are also preferably present in lyophilisedform. Before use, they must be resuspended in an appropriate solvent,for example double distilled water.

The following Examples are given for the purpose of illustrating thepresent invention:

EXAMPLE 1 Determination of the Total Number of T-Lymphocytes 1.Preparation of the Solutions

a) Standard solutions.

Latex particles based on methacrylate (prepared according to FederalRepublic of Germany Patent Specification No. 30 48 883) are activated atpH 5.0 with sodium periodate. The activated latex particles are dividedinto two halves. One half is coupled with non-specific sheep IgG and theother half with sheep IgG anti-mouse IgG. By mixing these two batches invarious defined proportions, three standard solutions are prepared whichcorrespond to a lymphocyte concentration of 0.45×10⁶, 1.56×10⁶ and3.36×10⁶ cells/ml. The solutions are lyophilised and, in this form, arestable for a comparatively long time. Before use, the standards areresuspended in double distilled water (calibration standard suspensionsa, b and c).

b) Wash buffer.

1.47 g. disodium hydrogen phosphate (Na₂ HPO₄. 2H₂ O), 0.27 g. sodiumdihydrogen phosphate (NaH₂ PO₄.H₂ O) and 8.77 g. sodium chloride aredissolved in double distilled water and made up to 1 liter with water.To this solution are added 0.2% w/v bovine serum albumin and 0.1% w/vsodium azide. For storage, it is lyophilised. Before use, thelyophilisate is resuspended in double distilled water.

c) Antibody conjugate.

Monoclonal antibody CD 6 (M-T411, characterised in detail in: P. Rieberet al., in Leucocyte typing (A. Bernard et al., ed.) 1984, page 303-311,pub. Springer Verlag, Berlin, N.Y.) is coupled with β-D galactosidaseand lyophilised. Per determination, there are used 0.025 U, dissolved inwash buffer.

d) Substrate solution.

60 mg. Chlorophenol red β-D-galactoside are dissolved in 30 ml.substrate buffer (100 mmol/liter HEPES buffer (pH 7.1), 2 mmol/litermagnesium aspartate, 1% w/v bovine serum albumin and 0.1% w/v sodiumazide).

2. Sample preparation.

A blood sample is mixed with ethylenediaminetetraacetic acid disodiumsalt (EDTA-Na₂.2H₂ O). 7.5 ml. of this fresh EDTA blood are mixed with15 ml. lysis buffer (155 mmol/liter ammonium chloride; 10 mmol/literpotassium hydrogen carbonate and 0.1 mmol/liter EDTA-Na₂). The mixtureis left to stand for 7 to 10 minutes at ambient temperature, it therebybeing carefully stirred up two or three times. Thereafter, it iscentrifuged for 10 minutes at 300 g. The supernatant with the lysederythrocytes is sucked off (Pasteur pipette, vacuum pump).

The leukocyte sediment is whirled up (Vortex mixer) and, for the lysisof residual erythrocytes, it is again resuspended in 2 ml. lysis buffer.The suspension is left to stand for 5 minutes, with occasional stirring.Thereafter, 4 ml. PBS (phosphate-buffered saline) solution (150mmol/liter sodium chloride and 10 mmol/liter sodium phosphate buffer (pH7.3)) is added thereto. It is again centrifuged for 10 minutes at 300 gand the supernatant is sucked off.

The cell sediment is whirled up and washed twice with, in each case, 5ml. PBS solution. After renewed centrifuging, the supernatants arecarefully sucked off. The sediment is whirled up and taken up with 2 ml.resuspension buffer (consisting of PBS solution, 0.2% w/v bovine serumalbumin, 1% w/v bovine serum immunoglobin and 0.1% w/v sodium azide.This cell suspension is immediately further worked up.

3. Carrying out of the measurement.

Wavelength: 578 nm

Temperature: ambient temperature (20°-25° C.)

Semimicrocuvette: 1 cm. layer thickness

Measurement volume: 1 ml; measurement against reagent blank

Into centrifuge tubelets with conical bottom are pipetted:

    ______________________________________                                                  standard                                                                      a     b         c       sample                                      ______________________________________                                        calibration 0.5 ml. 0.5 ml.   0.5 ml.                                                                             --                                        standard                                                                      suspensions                                                                   a/b/c                                                                         wash buffer 2.0 ml. 2.0 ml.   2.0 ml.                                                                             --                                        ______________________________________                                    

Centrifuging is carried out for 10 minutes at 300 g. The supernatantsare carefully sucked off and the sediments are whirled up and thenpipetted as follows:

    ______________________________________                                                   standard                                                                      a     b         c       sample                                     ______________________________________                                        wash buffer  0.2 ml. 0.2 ml.   0.2 ml.                                                                             --                                       cell suspension                                                                            --      --        --    0.2 ml.                                  (sample from 2)                                                               antibody     0.1 ml. 0.1 ml.   0.1 ml.                                                                             0.1 ml.                                  conjugate                                                                     ______________________________________                                    

After thorough mixing, incubation is carried out for 45 minutes atambient temperature (20°-25° C.) with vigorous shaking up at 15 minuteintervals. Thereafter, in each case, 2 ml. wash buffer are addedthereto, again mixed and centrifuged at 300 g for 10 minutes. Thesupernatants are carefully sucked off and the sediments whirled up. Theyare mixed several times with, in each case, 2 ml. wash buffer andcentrifuged at 300 g for 10 minutes. The supernatants are againcarefully sucked off and the sediments whirled up. Subsequently, theyare resuspended with, in each case, 1 ml. substrate solution(chlorophenol red β-galactoside). They are well mixed and incubated for30 minutes at ambient temperature (20°-25° C.), whereby, after 15minutes and 30 minutes, in each case they are vigorously shaken andcentrifuged for 5 minutes at 700 g. The extinctions of the clearsupernatants are measured against the substrate solution as reagentblank.

4. Evaluation.

With the help of the measured calibration standard values and thecorresponding concentrations, there is produced a calibration curve.From this is directly read off the cell value for the samplecorresponding to the measurement value.

EXAMPLE 2 Determination of the T-Helper Subpopulation

The preparation of the sample, the carrying out of the measurement andthe evaluation take place in the manner described in Example 1. However,as calibration standard suspensions a, b and c, there are usedsuspensions which correspond to a T-helper concentration of 0.20×10⁶,0.60×10⁶ and 2.08×10⁶ cells/ml. Furthermore, as antibody conjugate,there is used a conjugate of a monoclonal antibody CD 4 (M-T 151,characterised in more detail in v. supra), which is specificallydirected against surface antigens which are characteristic for theT-helper subpopulation, and β-galactosidase. 0.05 U Antibody areemployed for each determination.

From the measured calibration standard values, there can be produced acalibration line from which can be directly read off the cell values forthe sample corresponding to the measurement values.

EXAMPLE 3 Determination of the T-Suppressor Subpopulation

The sample preparation and the carrying out of the measurement iscarried out analogously to Example 1. However, as antibody conjugate,there is used a conjugate of β-galactosidase and an antibody which isspecifically directed to a surface antigen of the T-suppressor cells, CD8 (M-T 811, characterised in more detail v. supra). For eachdetermination, there are used 0.025 U of antibody. Furthermore, ascalibration standard suspensions a, b and c, there are used threesuspensions of different concentration which correspond to aT-suppressor concentration of 0.29×10⁶, 0.62×10⁶ and 1.17×10⁶ cells/ml.

The results obtained with the reagent according to the present inventionusing the ELISA process of the present invention correlate very wellwith values which are obtained by the previously usual, substantiallymore laborious method of mechanical counting (cf. FIG. 1, FIG. 2 andFIG. 3 of the accompanying drawings. In these:

FIG. 1 shows a method comparison FACS/process according to the presentinvention for total T-lymphocytes (t-pan);

FIG. 2 shows a method comparison FACS/process according to the presentinvention for T-helper subpopulation (t-helper); and

FIG. 3 shows a method comparison FACS/process according to the presentinvention for T-suppressor subpopulation (t-suppressor).

It will be understood that the specification and examples areillustrative but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

What is claimed is:
 1. A standard for the quantification of a cellpopulation or subpopulation, comprising:a mixture of particles having aknown concentration, which mixture has sedimentation behavior similar tothe sedimentation behavior of the population or subpopulation to bequantified, a first portion of said mixture of particles carrying anon-specific antibody and a second portion of said mixture of particlescarrying molecules directed against a labelled antibody whichspecifically binds to a cell surface antigen characteristic of the cellpopulation or subpopulation to be quantified, wherein said first portionand said second portion of said mixture are identical except for theantibody carried by the first portion and the molecules carried by thesecond portion.
 2. The standard of claim 1, wherein the particlesconsist of glass or synthetic resin.
 3. The standard of claim 2, whereinthe synthetic resin is based on methacrylate.
 4. The standard of claim1, wherein the molecules carried by the second portion of said mixtureof particles are selected from the group consisting of (i) proteinswhich possess the same antigenicity as the cell surface antigencharacteristic of the population or subpopulation to be quantified; (ii)anti-antibodies which specifically bind to the antibody portion of thelabelled antibody and (iii) antibodies which specifically bind to thelabel portion of the labelled antibody.
 5. The standard of claim 1,wherein said particles have a diameter of from 1 to 20 μm.
 6. Thestandard of claim 1, wherein said particles have a diameter of from 7 to13 μm.
 7. The standard of claim 1, wherein the molecule carried by thesecond portion of said mixture of particles is an antibody whichspecifically binds to the Fc portion of the labelled antibody.